Development of a novel apigenin prodrug programmed for alkaline-phosphatase instructed self-inhibition to combat cancer.

Elevated levels of alkaline phosphatase (ALP) in the tumor microenvironment (TME) are a hallmark of cancer progression and thus inhibition of ALP could serve as an effective approach against cancer. Herein, we developed a novel prodrug approach to tackle cancer that bears self-inhibiting alkaline phosphatase-responsiveness properties that can enhance at the same time the solubility of the parent compound. To probe this novel concept, we selected apigenin as the cytotoxic agent since we first unveiled, that it directly interacts and inhibits ALP activity. Consequently, we rationally designed and synthesized, using a self-immolative linker, an ALP responsive apigenin-based phosphate prodrug, phospho-apigenin. Phospho-apigenin markedly increased the stability of the parent compound apigenin. Furthermore, the prodrug exhibited enhanced antiproliferative effect in malignant cells with elevated ALP levels, compared to apigenin. This recorded potency of the developed prodrug was further confirmed in vivo where phospho-apigenin significantly suppressed by 52.8% the growth of PC-3 xenograft tumors.Communicated by Ramaswamy H. Sarma.

Using Sister Chromatid Exchange Assay to Detect Homologous Recombination Deficiency in Epigenetically Deregulated Urothelial Carcinoma Cells.

Sister chromatid exchange (SCE) is the process of exchanging regions between two sister chromatids during DNA replication. Exchanges between replicated chromatids and their sisters can be visualized in cells when DNA synthesis in one chromatid is labelled by 5-bromo-2'-deoxyuridine (BrdU). Homologous recombination (HR) is considered as the principal mechanism responsible for the sister chromatid exchange (SCE) upon replication fork collapse, and therefore SCE frequency upon genotoxic conditions reflects the capacity of HR repair to respond to replication stress. During tumorigenesis, inactivating mutations or altered transcriptome can affect a plethora of epigenetic factors that participate in DNA repair processes, and there are an increasing number of reports which demonstrate a link between epigenetic deregulation in cancer and homologous recombination deficiency (HRD). Therefore, the SCE assay can provide valuable information regarding the HR functionality in tumors with epigenetic deficiencies. In this chapter, we provide a method to visualize SCEs. The technique outlined below is characterized by high sensitivity and specificity and has been successfully applied to human bladder cancer cell lines. In this context, this technique could be used to characterize the dynamics of HR repair in tumors with deregulated epigenome.

Cancer Evolution in Precision Medicine Era.

Recent advances in our understanding of cancer, driven mainly by the emergence of new technologies have highlighted that heterogeneity shapes not only the genetic profile of tumors but also their epigenetic and gene expression profile [...].

DNA Methylation Analysis in Plasma Cell-Free DNA and Paired CTCs of NSCLC Patients before and after Osimertinib Treatment.

Osimertinib has been an effective second-line treatment in EGFR mutant NSCLC patients; however, resistance inevitably occurs. DNA methylation has been previously implicated in NSCLC progression and often in therapy resistance, however its distinct role in osimertinib resistance is not elucidated as yet. In the present study, we directly compared DNA methylation of nine selected genes (RASSF1A, RASSF10, APC, WIF-1, BRMS1, SLFN11, RARß, SHISA3, and FOXA1) in plasma-cfDNA and paired CTCs of NSCLC patients who were longitudinally monitored during osimertinib treatment. Peripheral blood (PB) from 42 NSCLC patients was obtained at two time points: (a) baseline: before treatment with osimertinib and (b) at progression of disease (PD). DNA methylation of the selected genes was detected in plasma-cfDNA (n = 80) and in paired CTCs (n = 74). Direct comparison of DNA methylation of six genes between plasma-cfDNA and paired CTC samples (n = 70) revealed a low concordance, indicating that CTCs and cfDNA give complementary information. DNA methylation analysis of plasma-cfDNA and CTCs indicated that when at least one of these genes was methylated there was a statistically significant increase at PD compared to baseline (p = 0.031). For the first time, DNA methylation analysis in plasma-cfDNA and paired CTCs of NSCLC patients during osimertinib therapy indicated that DNA methylation of these genes could be a possible resistance mechanism.

USP44 Promoter Methylation in Plasma Cell-Free DNA in Prostate Cancer.

Liquid biopsy provides real-time monitoring of tumor evolution and response to therapy through analysis of circulating tumor cells (CTCs) and plasma-circulating tumor DNA (ctDNA). USP44 is a critical gene which plays an important role in cell proliferation; however, its accurate role in other cellular networks is under research. USP44 promoter methylation has been so far reported in colorectal neoplasia and metastatic breast cancer. In this study, we examined for the first time USP44 promoter methylation in plasma cell-free DNA (cfDNA) of patients with prostate cancer (early stage n = 32, metastatic n = 39) and 10 healthy donors (HD). USP44 promoter methylation was detected in plasma cell-free DNA by a newly developed highly specific and sensitive real-time MSP method. Our findings indicate that USP44 promoter is methylated in plasma cell-free DNA of metastatic prostate cancer patients and that detection of USP44 promoter methylation is significantly associated with overall survival (OS) (p = 0.008). We report for the first time that detection of USP44 promoter methylation in plasma cell free DNA provides significant prognostic information in metastatic prostate cancer.

HDAC Inhibitors: Dissecting Mechanisms of Action to Counter Tumor Heterogeneity.

Intra-tumoral heterogeneity presents a major obstacle to cancer therapeutics, including conventional chemotherapy, immunotherapy, and targeted therapies. Stochastic events such as mutations, chromosomal aberrations, and epigenetic dysregulation, as well as micro-environmental selection pressures related to nutrient and oxygen availability, immune infiltration, and immunoediting processes can drive immense phenotypic variability in tumor cells. Here, we discuss how histone deacetylase inhibitors, a prominent class of epigenetic drugs, can be leveraged to counter tumor heterogeneity. We examine their effects on cellular processes that contribute to heterogeneity and provide insights on their mechanisms of action that could assist in the development of future therapeutic approaches.

Prognostic Significance of SLFN11 Methylation in Plasma Cell-Free DNA in Advanced High-Grade Serous Ovarian Cancer.

BACKGROUND: Epigenetic alterations in ctDNA are highly promising as a source of novel potential liquid biopsy biomarkers and comprise a very promising liquid biopsy approach in ovarian cancer, for early diagnosis, prognosis and response to treatment. METHODS: In the present study, we examined the methylation status of six gene promoters (BRCA1, CST6, MGMT, RASSF10, SLFN11 and USP44) in high-grade serous ovarian cancer (HGSOC). We evaluated the prognostic significance of DNA methylation of these six gene promoters in primary tumors (FFPEs) and plasma cfDNA samples from patients with early, advanced and metastatic HGSOC. RESULTS: We report for the first time that the DNA methylation of SLFN11 in plasma cfDNA was significantly correlated with worse PFS (p = 0.045) in advanced stage HGSOC. CONCLUSIONS: Our results strongly indicate that SLFN11 epigenetic inactivation could be a predictor of resistance to platinum drugs in ovarian cancer. Our results should be further validated in studies based on a larger cohort of patients, in order to further explore whether the DNA methylation of SLFN11 promoter could serve as a potential prognostic DNA methylation biomarker and a predictor of resistance to platinum-based chemotherapy in ovarian cancer.

Monitoring of Active Notch Signaling in Mouse Bladder Urothelium.

Notch signaling plays a crucial role in differentiation and homeostasis in a wide variety of epithelia. The tumor suppressor role of Notch in bladder urothelium is well accepted as the inactivation of this pathway due to damaging mutations in its components is associated with neoplastic transformation. Monitoring Notch signaling is therefore critical to understand how the deregulation of cell-cell communication can lead to differentiation loss and carcinogenesis. In this chapter, we provide a method to visualize active Notch signaling by the detection of the nuclear levels of Notch intracellular domain in mouse urothelium. The technique outlined below is characterized by high sensitivity and specificity and has been successfully applied to human tumor specimens. In this context, this technique could be used to characterize the molecular profile of Notch-deficient tumors and analyze the clonal expansion dynamics and the heterogeneity patterns of Notch inactivation.

Exploring the Eco-Evolutionary Dynamics of Tumor Subclones.

Mutational processes constantly shape the cancer genome and defects in DNA repair pathways of tumor cells facilitate the accumulation of genomic alterations [...].

Mitonuclear Interactions in the Maintenance of Mitochondrial Integrity.

In eukaryotic cells, mitochondria originated in an α-proteobacterial endosymbiont. Although these organelles harbor their own genome, the large majority of genes, originally encoded in the endosymbiont, were either lost or transferred to the nucleus. As a consequence, mitochondria have become semi-autonomous and most of their processes require the import of nuclear-encoded components to be functional. Therefore, the mitochondrial-specific translation has evolved to be coordinated by mitonuclear interactions to respond to the energetic demands of the cell, acquiring unique and mosaic features. However, mitochondrial-DNA-encoded genes are essential for the assembly of the respiratory chain complexes. Impaired mitochondrial function due to oxidative damage and mutations has been associated with numerous human pathologies, the aging process, and cancer. In this review, we highlight the unique features of mitochondrial protein synthesis and provide a comprehensive insight into the mitonuclear crosstalk and its co-evolution, as well as the vulnerabilities of the animal mitochondrial genome.

TP53 mutational landscape of metastatic head and neck cancer reveals patterns of mutation selection.

BACKGROUND: Head and neck squamous cell carcinoma is a heterogeneous disease with respect to the anatomic site of the primary tumor. On the other hand, it is highly recurrent, and once metastatic, it is associated with poor prognosis. TP53 is the most commonly mutated gene in primary disease. TP53 mutations occur in different structural elements of the protein while the biological outcome can be diverse. METHODS: Here we aimed to find differences in the mutation profile of TP53 in primary and metastatic disease and the impact of TP53 mutations in metastasis, specific copy number alterations, tumor mutation burden and response to immune checkpoint inhibitors. Somatic mutation and clinical data for 512 primary and 134 metastatic biopsies were studied. FINDINGS: Overall TP53 mutation frequency is significantly lower in metastases compared to primary tumors. One the other hand, missense mutations in the DNA binding region are significantly enriched in metastases and are associated with a common fragile site in chromosome 11, leading to amplification and overexpression of genes with established role in metastasis. Finally, TP53 mutations are associated with higher TMB score in metastatic but not primary tumors, and poorer response to immune checkpoint inhibitors for the latter. INTERPRETATION: TP53 mutations affect clinical and molecular aspects of head and neck tumorigenesis including metastasis, genetic alterations and therapeutic response. FUNDING: This work was supported by a Horizon 2020 grant (801347) to AK, and a Greek General Secretariat for Research and Technology and the Hellenic Foundation for Research and Innovation grant (472-EpiNotch) to TR.

Resolving DNA Damage: Epigenetic Regulation of DNA Repair.

Epigenetic research has rapidly evolved into a dynamic field of genome biology. Chromatin regulation has been proved to be an essential aspect for all genomic processes, including DNA repair. Chromatin structure is modified by enzymes and factors that deposit, erase, and interact with epigenetic marks such as DNA and histone modifications, as well as by complexes that remodel nucleosomes. In this review we discuss recent advances on how the chromatin state is modulated during this multi-step process of damage recognition, signaling, and repair. Moreover, we examine how chromatin is regulated when different pathways of DNA repair are utilized. Furthermore, we review additional modes of regulation of DNA repair, such as through the role of global and localized chromatin states in maintaining expression of DNA repair genes, as well as through the activity of epigenetic enzymes on non-nucleosome substrates. Finally, we discuss current and future applications of the mechanistic interplays between chromatin regulation and DNA repair in the context cancer treatment.

Targeting DNA repair in cancer: current state and novel approaches.

DNA damage response, DNA repair and genomic instability have been under study for their role in tumor initiation and progression for many years now. More recently, next-generation sequencing on cancer tissue from various patient cohorts have revealed mutations and epigenetic silencing of various genes encoding proteins with roles in these processes. These findings, together with the unequivocal role of DNA repair in therapeutic response, have fueled efforts toward the clinical exploitation of research findings. The successful example of PARP1/2 inhibitors has also supported these efforts and led to numerous preclinical and clinical trials with a large number of small molecules targeting various components involved in DNA repair singularly or in combination with other therapies. In this review, we focus on recent considerations related to DNA damage response and new DNA repair inhibition agents. We then discuss how immunotherapy can collaborate with these new drugs and how epigenetic drugs can rewire the activity of repair pathways and sensitize cancer cells to DNA repair inhibition therapies.

Unraveling UCA1 lncRNA prognostic utility in urothelial bladder cancer.

In the era of precision oncology, bladder cancer (BlCa) is characterized by generic patient management and lack of personalized prognosis and surveillance. Herein, we have studied the clinical significance of urothelial cancer associated 1 (UCA1) lncRNA in improving patients' risk stratification and prognosis. A screening cohort of 176 BlCa patients was used for UCA1 quantification. The Hedegaard et al. (n = 476) and The Cancer Genome Atlas (TCGA) provisional (n = 413) were analyzed as validation cohorts for non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC), respectively. Patients' survival outcome was assessed using recurrence and progression for NMIBC or death for MIBC as clinical endpoint events. Bootstrap analysis was performed for internal validation of Cox regression analysis, whereas the clinical benefit of disease prognosis was assessed by decision curve analysis. UCA1 was significantly overexpressed in bladder tumors compared with normal urothelium, which was confirmed only in the case of NMIBC. Interestingly, reduced expression of UCA1 was correlated with muscle-invasive disease as well as with tumors of higher stage and grade. UCA1 loss was strongly associated with higher risk of short-term relapse [hazard ratio (HR) = 1.974; P = 0.032] and progression to invasive stages (HR = 3.476; P = 0.023) in NMIBC. In this regard, Hedegaard et al. and TCGA validation cohorts confirmed the unfavorable prognostic nature of UCA1 loss in BlCa. Finally, prognosis prediction models integrating UCA1 underexpression and established clinical disease markers contributed to improved stratification specificity and superior clinical benefit for NMIBC prognosis. Underexpression of UCA1 correlates with worse disease outcome in NMIBC and contributes to superior prediction of disease early relapse and progression as well as improved patient stratification specificity.

The lysine-specific methyltransferase KMT2C/MLL3 regulates DNA repair components in cancer.

Genome-wide studies in tumor cells have indicated that chromatin-modifying proteins are commonly mutated in human cancers. The lysine-specific methyltransferase 2C (KMT2C/MLL3) is a putative tumor suppressor in several epithelia and in myeloid cells. Here, we show that downregulation of KMT2C in bladder cancer cells leads to extensive changes in the epigenetic status and the expression of DNA damage response and DNA repair genes. More specifically, cells with low KMT2C activity are deficient in homologous recombination-mediated double-strand break DNA repair. Consequently, these cells suffer from substantially higher endogenous DNA damage and genomic instability. Finally, these cells seem to rely heavily on PARP1/2 for DNA repair, and treatment with the PARP1/2 inhibitor olaparib leads to synthetic lethality, suggesting that cancer cells with low KMT2C expression are attractive targets for therapies with PARP1/2 inhibitors.

A new tumor suppressor role for the Notch pathway in bladder cancer.

The Notch signaling pathway controls cell fates through interactions between neighboring cells by positively or negatively affecting the processes of proliferation, differentiation and apoptosis in a context-dependent manner. This pathway has been implicated in human cancer as both an oncogene and a tumor suppressor. Here we report new inactivating mutations in Notch pathway components in over 40% of human bladder cancers examined. Bladder cancer is the fourth most commonly diagnosed malignancy in the male population of the United States. Thus far, driver mutations in fibroblast growth factor receptor 3 (FGFR3) and, less commonly, in RAS proteins have been identified. We show that Notch activation in bladder cancer cells suppresses proliferation both in vitro and in vivo by directly upregulating dual-specificity phosphatases (DUSPs), thus reducing the phosphorylation of ERK1 and ERK2 (ERK1/2). In mouse models, genetic inactivation of Notch signaling leads to Erk1/2 phosphorylation, resulting in tumorigenesis in the urinary tract. Collectively our findings show that loss of Notch activity is a driving event in urothelial cancer.

Markers of epithelial to mesenchymal transition in association with survival in head and neck squamous cell carcinoma (HNSCC).

BACKGROUND: Elucidating the molecular phenotype of cancers with high metastatic potential will facilitate the development of novel therapeutic approaches to the disease. Gene expression profiles link epithelial to mesenchymal transition (EMT) phenotype with high-risk HNSCC. We sought to determine the role of protein biomarkers of EMT in head and neck squamous carcinoma (HNSC) prognosis. METHODS: Protein expression analysis of EGFR, ß-catenin and E-cadherin was performed on a cohort of 102 patients with HNSCC recruited between 1992 and 2005 using automated quantitative protein analysis (AQUA). We evaluated associations with clinicopathological parameters and prognosis. RESULTS: There were 67 patients with primary squamous cell carcinoma of the head and neck in this cohort who met inclusion criteria and for whom we had complete E-cadherin, beta-catenin and EGFR expression data. High E-cadherin expressers had longer 5-year progression-free survival (PFS) compared to those with low E-cadherin (59.7% versus 40.6%, p = 0.04) and overall survival (OS) (69.6% versus 44.3%, p  = 0.05). Kaplan-Meier analysis showed that patients with low beta-catenin-expressing tumors trended toward worse 5-year PFS (p = 0.057). High EGFR expressers had inferior OS compared to low EGFR expressers (27.7% vs. 54%, p = 0.029). In the multivariable analysis context, E-cadherin remained an independent predictor of improved OS (HR = 0.204, 95% CI 0.043 to 0.972, p = 0.046) while EGFR trended towards significance for OS. CONCLUSIONS: The putative markers of EMT defined within a panel of HNSCC using AQUA are associated with tumors of poor prognosis.

Molecular mechanisms of HPV induced carcinogenesis in head and neck.

A growing subgroup of oropharyngeal cancers is initiated by infection with high-risk human papillomaviruses (HPVs). In parallel to mounting epidemiological evidence, solid experimental data support a causal association between HPV and a subset of oropharyngeal cancers. This Review summarizes the main events of the HPV life cycle, the functions of the viral oncoproteins, and the implications of HPV infection on their hosts, with an emphasis on carcinogenic mechanisms in oropharyngeal cancer. The demonstration that HPVs have a role in head and neck carcinogenesis has fuelled the expectation that HPV-targeted therapeutic strategies may prove curative in these cancers.